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Muscle actin cleaved by proteinase K: its polymerization and in vitro motility
S Higashi-Fujime1, M Suzuki, K Titani
1Department of Molecular Biology, Faculty of Science, Nagoya University, Aichi.
Abstract:
Skeletal muscle actin was lightly digested by proteinase K, which cleaved the peptide bond between Met-47 and Gly-48, producing a C-terminal 35 kDa fragment. Proteinase K-cleaved actin (proK-actin) did not polymerize into F-actin upon addition of salt. In the presence of phalloidin, however, it polymerized slowly into F-actin (proK-F-actin), indicating that the cleaved actin did not dissociate into the individual cleaved fragments but retained the global structure of actin. Electron microscopy showed that proK-F-actin had the typical double-stranded structure of a normal actin filament and formed the arrowhead structure when decorated with HMM. Heavy meromyosin ATPase was weakly activated by proK-F-actin: Vmax = 0.24 s-1, and Kapp = 2.8 microM, while Vmax = 7.6 s-1, and Kapp = 13 microM by F-actin. Correspondingly, in vitro this proK-F-actin slid very slowly on HMM attached to a glass surface at an average velocity of 0.47 microns/s, or 1/12 of that of intact F-actin. The fraction of sliding filaments was less than 50%. Assuming that the nonmotile filaments attached to HMM were not involved in ATPase activation, the sliding velocity correlated with the ATPase activity activated by proK-F-actin.
Insights
Proteinase K digestion of skeletal muscle actin creates a fragment that polymerizes slowly into filaments. These modified actin filaments exhibit reduced motor protein interaction and sliding velocity, impacting muscle function.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Actin polymerization is crucial for muscle contraction and cellular motility.
- Proteinase K is a serine protease used to study protein structure and function.
Purpose of the Study:
- To investigate the functional consequences of specific actin cleavage on polymerization and motor protein interaction.
- To characterize the properties of proteinase K-cleaved actin (proK-actin) filaments.
Main Methods:
- Limited digestion of skeletal muscle actin with proteinase K.
- Actin polymerization assays in the presence and absence of phalloidin.
- Electron microscopy for filament structure analysis.
- In vitro motility assays using heavy meromyosin (HMM) and ATPase activity measurements.
Main Results:
- Proteinase K cleaved actin at Met-47/Gly-48, yielding a 35 kDa fragment (proK-actin).
- proK-actin polymerized slowly into filaments (proK-F-actin) with phalloidin, retaining global actin structure.
- proK-F-actin filaments showed reduced HMM binding, ATPase activation (Vmax = 0.24 s⁻¹), and significantly slower sliding velocity (0.47 µm/s).
Conclusions:
- Specific cleavage of actin impairs its ability to interact with motor proteins like HMM.
- The reduced functional capacity of proK-actin filaments highlights the importance of the intact actin structure for efficient muscle contraction.
- These findings provide insights into the structure-function relationship of actin in muscle mechanics.